Method of producing rabies vaccine



Aug. 13, 1968 M. v. FERNANDES ETAL. 3,397,267

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MARIO V. FERNANDES ATTORNEYS United States Patent Oce 3,397,26' Patented Aug. 13, 1961 METHOD F PRODUCING RABIES VACCINE Mario V. Fernandes, Philadelphia, and Hilary Koprowski and Tadeusz J. Wiktor, Wynnewood, Pa., assignors to Research Corporation, New York, NX., a nonprofit corporation of New York Filed Sept. 21, 1964, Ser. No. 397,763 8 Claims. (Cl. 4124-89) ABSTRACT 0F THE DISCLOSURE Anti-rabies vaccines are made by the subculture passage adaptation of live rabies virus strains for propagation in normal human diploid cells.

This invention relates to an anti-rabies vaccine and more particularly to a novel vaccine advantageously adapted to provide excellent levels of immunity to virulent rabies virus in humans and Warm-blooded animals. The invention also relates to a method for preparing said anti-rabies vaccine.

Rabies is an extremely serious disease since, once it is contracted, no cure is available therefor and fatality rates are high. While the disease presently is not a major medical problem in the United States, indications are, however, that its incidence is on the increase. Moreover, contrary to the situation in the United States, in some countries abroad rabies is more prevalent and does constitute a signicant threat to the welfare of humans, as well as domestic animals. Accordingly, a reliable, safe, and effective anti-rabies vaccine, if available, would be highly valuable.

Attempts have been made previously t-o develop a vaccine which is effective against rabies Virus. The results of such efforts heretofore, however, have not been completely satisfactory in that the vaccines obtained either do not provide the requisite antigenic effects in terms of potency and/or duration and/or may even undesirably be characterized by an ability to produce unwanted side effects, sometimes permanent in nature, `due to damage to vital cells such as brain cells.

Accordingly, it is the primary object of the present invention to provide an anti-rabies vaccine which is superior to similar vaccines heretofore available.

It is an additional object of the present invention to provide an anti-rabies vaccine which is characterized by the ability to provide effective levels of immunization to humans and other wann-blooded animals against infection by rabies virus.

Still another object of the present invention is to provide a method for preparing said highly advantageous anti-rabies vaccine.

Broadly described, the present invention provides an anti-rabies vaccine comprising a rabies virus strain selected from the group consisting of live rabies Virus strains adapted for propagation growth in normal human diploid cells and inactivated species of said adapted live rabies virus strains.

The present invention further provides a method for producing an effective anti-rabies vaccine which corr prises growing in a sterile culture of normal human di; loid cells a strain of live rabies virus capable of infectin less than about 10% of said human diploid cells in sai culture and maintaining at least a portion of the resultar grown virus through a series of subculture passages i separate non-infected sterile cultures of the normal hl man diploid cells by direct transfer of infected huma diploid cells from a preceding passage in said series t provide a live rabies virus strain adapted to infect at leaf about yof the cells in a non-infected Sterile cultur of the normal human diploid cells.

In accordance with the present invention, the terr adapted, when employed to describe the growth chai actestics of the live rabies virus strains of the inventio in normal, i.e., non-infected, human diploid cells, is ir tended to refer to rabies virus strains produced by th above-described method which further are characterize by the ability to be propagated in fresh sterile cultures o the normal human diploid cells by a transfer of cell-fre medium from a human diploid cell culture infected there with. This characteristic of the live rabies virus strain employed in the vaccines `of the invention advantageousl distinguishes them from non-adapted rabies viru strains.

The human diploid cell tissue culture systems used i: the present method are known and available. A specifi example `of such culture systems is the human diploid cel strain culture described in Exper. Cell Res., 25, page 595 Propagation of the tissue culture systems in vitro ern ployed in the present method suitably may be carried ou by any of the standard methods described in the prio art. Specific examples of such propagation techniques ar disclosed in Exper. Cell Res., 25, page 595; Virology, 1 page 147; and J. Cell Biol., 17, page 299. The tissu culture systems, for example, usually comprise Eagle basal medium in Earles balanced salt solution supple mented with inactivated calf serum and containing i sterilizing amount of an antibiotic such as penicillin streptomycin, aureomycin, or mixtures thereof, the sys tem being buffered at a pH of about 6.8-8.5 with a con ventional biological buffering agent, such as an alkal metal, e.g., sodium bicarbonate, carbonate, Ior hydrogei phosphate.

Live rabies virus strains heretofore available were non adapted for growth in normal human diploid cells an( constitute the starting virus employed in the presen method. Such non-adapted rabies virus are readily avail able. Specific examples lof such non-adapted strains o rabies virus include (a) a standard challenge rabies viru strain (CVS), a xed rabies virus strain propagated il mouse brains and available at the National Institutes o Health, Bethesda, Maryland, (b) the Pittman-Moor' rabies virus strain (PM), a fixed rabies virus strain propa gated in rabbit brains and available at the National Insti tutes of Health, and (c) Flury high egg passage virus straii (HEP), a chicken embryo adapted rabies virus strain a the 225th egg passage, described in Bull. WHO, 10, pag 799. The HEP rabies virus strain constitutes the preferre( starting strain employed in preparing the vaccines of th present invention.

In accordance with the present method, a sample of non-adapted live virulent rabies virus strain, usually 1 the form of an aqueous virus pool consisting of from bout a to 25% suspension of infected tissue, e.g., [EP in chicken embryo, in about 40% to 60% normal alf serum in distilled water, is mixed with the human iploid cell tissue culture medium, and the resultant rabies irus-containing culture mediuml is incubated to allow le virus to infect the cells. The mixing ratio of rabies irus to human diploid cells in the tissue culture medium referably is in the range of from about l 105 to about X 10B LD50 of virus per 1 105 cells of the human dipid cell tissue. The incubation for the initial steps as 'ell as the succeeding subcultivation passage steps, hereiafter described, preferably is carrier out at a temperture in the range of from about 30 to about 40 C.

The incubation of the treated tissue culture medium referably is allowed to stand until the tissue cell culture ecomes coniiuent, usually in about 3 to 4 days, and no ppreciable further infection of the cells is observed.

At the end of the initial step of the process at least a ortion of the resultant rabies virus grown in the in- :cted tissue culture system is transferred to a fresh hu- 1an diploid cell culture system. The transfer suitably is arried out by a transfer of virus-infected cells from the iitial culture system with the mixing of same with fresh :erile human diploid cells while maintaining the virus live. In the transfer step, in instances wherein an agent L employed to detach the infected cells to be transferred fom the remainder of the culture and a support on which 1e cultivation is carried out, the transfer aid preferably a proteolytic enzyme such as trypsin. The mixture of 1e virus-infected cells obtained from the first stage and 1e fresh human diploid cell culture medium then is inubated, as described in relation tothe first stage, to -allow 1e Arabies virus to infect and grow in the resultant cul- In accordance with the invention, the aforedescribed 'ansfer of the virus-infected human diploid cells to a icceeding subcultivation stage, mixing of transferred in- :cted cells with fresh human diploid cells in a tissue cul- 1re medium thereof and incubation of the resultant mix- 1re of infected and fresh cells to infect and grow the trans- :rred rabies virus strain in the fresh tissue culture 'are arried out over a requisite number of stages of subculvations to provide a rabies virus strain adapted for rowth propagation in human diploid cells. Rabies virus :rains so poduced and which are so adapted have the baracteristic of the strain being able to be propagated irough succeeding serial stages by transfer merely of :ll-free medium containing the virus strain from an iu- :cted culture to a fresh human diploid cell culture wherel at least about 90% of the cells are infected thereby.

The actual number of culture passages required in :lapting rabies virus to growth in human diploid cells deends principally upon the particular starting rabies virus rain. The number of serial culture passages generally tilized is at least about 10. In preferred embodiments of 1e invention employing human diploid cell culture sys- :m's and starting with non-adapted HEP or CVS rabies irus strains, the requisite number of serial culture pasiges to provide an adapted virus strain is in the range f from about 45 to about 50.

The adapted rabies virus strain may be employed as live vaccine, or it may be killed by any standard virus lactivation procedure using phenol, beta-propiolactone nd the like inactivating agents and employed as a vacne in inactivated form.

The invention having been described above in detail, 1e following examples are given to show further specific nbodiments thereof. It will be understood the examples re given merely for illustration purposes and not by vay of limitation.

The basic materials employed in the work represented y the data appearing in the examples are the following:

Virus strains (l) Standard challenge virus strain (CVS), a fixed rabies virus propagated in mouse brain, available at the National Institutes of Health, Bethesda, Maryland.

(2) Pittman-Moore virus strain (PM), a fixed rabies virus propagated in rabbit brain, available at the National Institutes of Health.

(3) Flury high egg passage virus strain (HEP), a chicken embryo adapted rabies virus at the 225th egg passage, described in Bull. WHO, 10, page 799.

Pools of the above rabies virus str-ains consisting of la 20% suspension of the infected tissue in 50% normal calf serum in distilled H2O were kept at 70 C.

(4) An attenuated poliovirus type 1 strain (CHAT) grown in a human diploid cell strain.

(5) Eastern equine encephalomyelitis virus (EEE) grown in primary chicken fibroblasts.

The tissue culture system used was human diploid cell strain (HDCS), derived from embryonic lung as described in Exper. Cell Res., 25, page 595.

The tissue culture system was propagated in vitro by the method described in Exper. Cell Res., 25, 595. Eagles basal medium in Earles balanced salt solution supplemented with 10% inactivated calf serum and containing 25 ml. of 5.6% sodium bicarbonate, units of penicillin and 105 ng. of streptomycin or 5 104 lig. of aureomycin per liter was used throughout. Cultures of HDCS were trans ferred twice weekly, using a 0.25% solution of tryspin in phosphate buffered saline supplemented with the antibiotics described above.

Example I About 1 106 LD50 of HEP virus were mixed with about 106 of dispersed HDCS cells in suspension. The resultant mixture was plated into culture bottles and incubated at about 37 C. for about 3 days. At the end of the incubation period the infectivity of the culture was determined by the immunofluorescent antibody staining and titrating technique described in I. Exper. Med., 91, page 1, as modified in Bull. WHO, 20, page 579, using antirabliesglobulin conjugate available at the National Rabies Laboratory, Atlanta, Georgia. The virus-infected culture prelparation was also examined for cytopathic effects and coverslip preparations were also st-ained with May-Greenwald-Giemsa, 'after fixation for 20 minutes in Bouins fixative, in order to determine the presence of inclusion bodies an-d confirm the destructive effect of the virus on the cell. The presence of HEP virus was determined by intracerebral inoculation (.03 ml.) of 4-day-old Swiss stock mice. For titration purposes, tenfold dilutions of the virus were inoculated into 5 to 10 animals per dilution. The mice were observed for 21 days and the endpoint was calculated according to the method described in Amer. J. Hyg., 27, 493. Medium from primary infected cultures was used to infect suspensions of the new cells. Infected cells were detached from glass by action of trypsin, resuspended in fresh medium, divided into two fractions and transferred to tissue culture vessels of twice the original surface. The fluorescing (staining) cell, destruction and virus presence tests were repeated on the resultant infected cultures after the described incubation period. Transfer of the cell-free medium lwas repeated as indicated in Table 1. The division and transfer of virus containing infected cells could be carried for 20 passages before there was total cell lysis. It was then necessary, lin order to maintain serial virus passage, to mix infected cells with a suspension of new non-in-fected homologous cells as indicated in Table 1. The characteristics of the virus strains produced were determined as described above. The results of the tests are set forth below in Table 1.

TABLE 1.-ADAPTATION OF HEP RABIES VIRUS TO GROWTH IN HDCS Procedures for Virus Transfers As shown in Table l, HEP rabies virus could infect transfer passage in order to maintain the virus in the tissu HDCS cells either las monolayers or dispersed cell cultures, but serial transfer of either the medium or the cell extracts in a homologous tissue culture system resulted in a gradual decrease of infectivity which was finally lost after 7 passages. The results obtained in such transfer methods through the irnmunouorescent staining of the infected tisue culture systems indicated that very few cells produced rabies virus antigen at any given time after infection with medium from the preceding culture. The small amounts of infective virus apparently made it impossible to propagate the virus either by transferring the medium or the cell-free extract from infected cultures.

Following'lysis of the infected cultures at the 20th passage level, attempts Were again made as shown in Table 1 to propagate the virus by transfer of cell-free media. Table 1 shows, however, that these attempts were unsuccessful and portions of the infected culture had to be mixed with non-infected homologous cells at each culture system. Using Vthis technique, fluorescence of al cells was observed on the third day after cell transfer ant HEP strains of rabies virus could be maintained for a1 unlimited number of passages. Table l also shows tha after maintaining the HEP strain for 47 tissue cultur passages using the cell-mixing transfer technique, th attempt to propagate the virus by transfer of cell-free tis sue culture medium was successful and it was possible t1 maintain virus in serial passages.

Example II The basic procedure of Example I was repeated with tht exceptions of substituting CVS rabies virus strain for th HEP rabies virus strain and employing 4 to 5-Week-oh Swiss mice of the same stock in the virus presence tests The transfer techniques and results thereof are set fortl in Table 2.

TABLE 2.--ADAPTATION OF CVS BABIES VIRUS TO GROWTH IN HDCS Procedures for Virus Transfers CPE) or Fluorcscing (IF). AMI-Adult mice LD50/0.03 ml. of mediul Table 2 reveals also that CVS rabies virus may be dapted to HDCS by means of the cell-mixing techique of Example I after 47 tissue culture passages and 1e resultant adapted CVS strain may be propagated therefter by transfer of cell-free tissue culture medium.

Example III In order to verify the identity of adapted rabies virus iter its serial passages in HDCS cells, serial tenfold ilutions of HEP before and after adaptation to HDCS as er Example I were mixed with normal and anti-rabies rra, respectively, incubated for 1 hour at 37 C. and then ijected intracerebrally into baby mice. The test was per- )rmed in young mice injected intracerebrally according i the standard method described in Laboratory Techiques in Rabies, Monograph Series, No. 23, WHO, 1954. knti-rabies sera were obtained from three sources: (a) ried anti-rabies gamma globulin prepared in horses at 1e Metchnikoif Research Institute, Moscow; (b) antiibies serum prepared in horses at the Pasteur Institute, aris; and (c) human anti-rabies serum from a man who ad been immunized by the classical Pasteur treatment nd had, in addition, received 3 booster inoculations of 'lury HEP virus. The results, presented in Table 3 below, idicate clearly that the HDCS-adapted virus was neu- 'alized by anti-rabies serum. Similar results were obiined when CVS and PM virus strains were tested.

ABLE 3.-SERONEUTRALIZATION OF HEP STRAIN OF VIRUS BEFORE AND AFTER ADAPTATION TO HDCS IN BABY MICE Morality Ratio of Mice Inoculated with Mixtures of Serum and Dlutions of Virus* (Dilutions) Seruin Used lor Neutralization A 10I 102 103 104 105 10e assage Level:

0 {Normal 7/7 9/9 9/9 7/7 3/7 0/6 Anti-Rabies. 0/7 0/7 0/8 0/6 49 {Normal ..8/8 8/8 8/8 8/8 8/8 4/8 0/8 Anti-Rabies 6/8 0/8 0/8 0/8 0/8 0/8 Noniinator=No, oi mice who died after inoculation. Denomi- Ltor=No. of mice inoculated. A Undiluted.

Example IV The data in Table 4 indicate that there was no direct correlation between infectivity of the virus for baby mice and the development of cytopathic effect and suggest that in the course of continuous propagation in the human diploid cell strain (HDCS), HEP virus particles were selected which had greater anity for the tissue culture system in which they were propagated.

Example V To further determine the modication of the properties of rabies virus adapted in accordance with the present invention, the pathogenicity of HEP rabies virus before adaptation to HDCS and after adaptation thereto as per Example I was compared on monkeys. In the tests 2 Rhesus monkeys weighing about 6 to 8 pounds were injected with about 0.25 ml. of 2 dilutions of egg-adapted HEP virus before its `adaptation to HDCS, became sick (on the 12th 4day after inoculation) and were sacriced when moribund 2 days later. At the same time 2 other monkeys of the same type injected intracerebrally with higher concentrations of HEP virus obtained at the 47th passage level in HDCS in accordance with Example I showed no signs of illness during a 6-month observation period. Groups of 8 and 2 monkeys of the same type also were injected intracerebrally with 103-5 and 105-5 baby mouse LD50 (0.25 ml.) of the HEP virus at the 47th passage in HDCS. None of the animals showed signs of sickness during a 60-day observation period. The results of the tests are set forth in Table 5.

TABLE 5.-RESULTS OF INTRACEREBRAL INOCULATION OF MONKEYS WITH HEP VIRUS BEFORE AND AFTER ADAPTATION TO HDCS Infeetivity Ratio of Monkeys Injected Intracerebl'ally with Baby Mouse LD50* (login) Experiment No.. 6. 5 5. 5 4. 5 3. 5

Passage Level:

*Nominator=No. of monkeys who died after inoculation. Den0minator=No. o inoculated monkeys with no signs of illness.

Table 5 shows that adaptation to HDCS not only did not increase pathogenicity of the HEP virus sfor primates, but that the adapted virus apparently lost its lethal properties for monkeys when injected directly into the central nervous system.

Example VI ABLE ir-CYTOPATHIC EFFECT AND IMMUNOFLUORESCENCE OF HEP STRAIN OF RABIES VIRUS-AT DIFFERENT PASSAGE LEVELS Code percent of cells destroyed (CPE) or Fluoreseing (IF) as recorde oul te 12th day then injected intracerebrally into young adult mice. The results of the tests are set forth in Table 6.

Mortality Ratio of Adult Mice Inoculated with Baby Dilutions of Mouse Brain Infected with Virus Baby Mouse HEP Dilutions Brain Inocula Egg-Adapted HD CS- Adapted NSA RSAA NS RS *Nominator=No. of mice who died after inoculation of baby mice brain tissue infected with HEP. Denominator=No. of adult mice inoculated. A NS=Normal Serum. AARS=Serum with Rabies Antibodies.

As can be seen from Table 6r, lonly in one instance did passage of HDCS-adapted HEP produce a rabies virus population `which seemed to become pathogenic for adult mice; whereas, rabies virus pathogenic -for adult mice has been irecovered from the brain tissue of lbaby mice injected with all dilutions (except 101) of egg-adapted HEP.

Example VII Example VIII The resistance of rabies-infected HDCS cultures to reinfection with homologous and heterologous viruses was investigated. Because HEP virus is not pathogenic for adult mice injected intracerebrally it was possible to study interference in HDCS infected with this strain of rabies Virus and re-exposed to another rabies strain, such as PM, which retained its pathogenicity for adult mice.

Cultures of HDCS infected with the HEP strain of virus at the 14th passage level as per Example I and showing 100% uorescing cells were trypsinized, counted and exposed, in suspension, to the PM strain of virus. The procedure was repeated for non-infected HDCS. After adsorption for 2 hours at 37 C. Vwith constant agitation, the cells were centrifuged, washed 3 times With new medium and planted in Petri dishes containing coverslips. After and days of incubation, respectively, the coverslips were stained to determine immunofluorescence and the supernatant Imedium was titrated in adult mice about 4 to 5 -weeks old by intracerebral inoculation to determine the presence of PM virus.

The end point of the titration was determined as described above with respect to HEP titrations. The result of the tests are set forth in Table 7.

TABLE 7,-RESISTANCE OF HEP-INFECTED HDCS T( RE-INFEC`TION WITH PM STRAIN OF RABIES Percent Cells Showing IFAS LD50 Titel' o. HDCS Cultures (Days after exposure to PM virus) Medium in Infected With- Adult Mice IIE P A 0 Nonc 1 20 103. 6

* All cells fluorcscing because of infection with HEP. A At 14th passage level in HDCS.

The results shown in Table 7 indicate that the Plv challenge virus apparently could not multiply in the HEP infected cells, although it did infect and multiply ir HDCS controls.

To determine the resistance of HDCS-infected culture to challenge with heterologous virus, one day after mono layers had formed in the cultures, several HEP, CVS and PM rabies-infected HDCS cultures prepared as pe Examples I and II and containing uorescing cell: and control HDCS cultures were exposed to chalrlengi with serial tenfold dilutions of either the polio or EEI virus described above containing a maximum of 5 10 plaque forming units and 5 l03 plaque forming units respectively. After one hour of adsorption the inoculun of the cultures was removed and, when the cultures hat been washed 3 times with phosphate buffered saline, agar overlay was added. The cultures were incubated for E days at 37 C. and then stained with neutral red solutior at a 1:10,()00 concentration in order to determine the number of plaques of either polio or EEE virus. Three Petri dishes were used for each dilution of challenge virus. The results of the tests are set forth in Table 8.

TABLE s.-RESIs'rANoE oF RABIESJNFECTED HDCS T( CHALLENGE WITH POLIOVIRUS AND EASTERN EQUINI ENCEPHALOMYELITIS (EEE) HDCS Average of Plaques Observez Infected After Infection with Challengi with Virus Dilutions Challenge Virus Rabies Virus 10-3 10-4 10-5 l0-G 10-7 Strains* R N R N R N R N R N CVS 0 0 0 O 0 Polio PM 0 C 0 C 0 C 0 53 0 3 Il 0 0` 0 0l 0 0 ol o o EEE --irrEP ci@ 01C oi 014 *Passage level in HDCS.' CVS at 49th; PM at 10th; HEP at 14th.

R=Rabiesinfected. N=Noninlected controls. C=Complete cell lysis.

The results of challenge shown in Table 8 show complete resistance of rabies-infected HDCS cultures to infection with the poliovirus and EEE virus.

Example IX TABLE 9.-RESULTS OF PROTECTION TEST* IN ADULT MICE 'USING LIVE RABIES VACCINE PREPARED FROM IIDCS-ADAPTED HEP Vaccine Titration of Challenge Virus (CVS) B aby mouse LD50 in LD50 in Passage D50 in vaccinated control Protection level inoculated mice mice index (logro) (logro) (logra) I3 6. Ol) 4. 15 6. 50 224 47 7. 00 1. 63 (i. 50 74, 000

*Two intraperitoneal injections ol vaccine given at 7-day intervals. Intracerebral challenge 21 days alter the lirst injection.

1 1 1 2 The results of the challenge inoculation set forth in a final concentration of 0.5% and incubation at 37 C. Fable 9 indicate that both preparations of the HEP virus for 48 hours or by treatment with beta propiolactone at mmunized mice against challenge with the greater degree a final concentration of %000 and incubation at 4 C. for )f resistance being shown by mice injected with the virus 24 hours. Twenty S-Week-old mice were inoculated init the 47th passage level. 5 tracerebrally with undiluted vaccine and tested for the Example X absence of infective virus. Intracerebral inoculation of twenty 5week-old mice with undiluted inactivated vac- The immunizing characteristics of the HDCS-adapted cines failed to reveal the presence of infectious virus. 'abies virus of the invention were further demonstrated Tests for the presence of infectious virus were carried out my immediately challenging the hamsters and guinea pigs, `as per Example II. The 5 lots of vaccine were then subvhich were living following their treatment described in m mitted to a standard Habel test, described in Laboratory Example VII, with a lethal dose of CVS virus as per the Techniques in Rabies, Monograph Series, No. 23, WHO, irocedure of Example IX. The results of these tests shown 1954. The results f the test are set forth in Table 11.

TABLE l1.-EFFECT OF TWO INACTIVATING AGENTS ON POTENCY OF BABIES VIRUS PREPARED FROM CVS AND PINI STRAINS GROWN IN HDCS (HABEL TEST) Vaccine Titi'ation of Challenge Virus (CVS) Passage LDsutiterin Inactivat- LD50 in LD50 in Virus level in mice before ing vaccinated control Protection strain HDCS inactivation agent micc(logi0) mice index (login) (101510) 13 4.12 Pheno1 4.90 0.20 23 i 3.00 do 4.98 6.26 is 9 .50 BPL 1.11 5.63 30,380 47 4.30 BPL 0. 74 5.63 70,750 12 3. a0 BPL 1.57 5. 03 11,500

BPL=Beta propiolactone.

n the lligure indicate that the HEP-inoculated animals be- Table l1 shows that, whereas the potency of the 2 CVS :ame resistant to CVS virus. preparations inactivated with phenol was very low, the immunizing capacity of the 3 vaccines prepared from Exampl (I CVS and PM strains and inactivated by BPL was Three Rhesus monkeys weighing 6 to 8 pounds were 35 extremely high, njected intramuscularly twice at 4-day intervals with un- The work on which this application was based was liluted tissue culture fluids, representing the 47th passage financed in whole 0r part hy the Public Health Service, )f HEP virus in HDCS as per Example I and containing We claim; l 107 baby mouse LD50 For COmPallsOn, aflhef gl'PuP 1. A method for producing an effective fanti-rabies vac- )f 3 Rhesus mfmkeys 0f the same s tOCk Was lnleted slim" 40 cine which comprises growing -in a sterile culture of norafly Wlth a Gluck embryo PfePal'alOn 0f HEP Vlflls PUOI mal human diploid cells a strain of live rabies virus capa- 0 lts adaptatlon t0 HDCS- Sera Were Obt'amed from the ble of infecting less than about 10% of said human dipnoculated animals 14 days after the rst virus inoculation loid cells in Said culture and maintaining at least .a porind were submitted to the neutralizationl test described in tion of the resultant grown Vil-ns through a series of Sub- Example III. rShe results of the tests are listed in Table 10. culture passages in separate non infeoted sterile cultures ALisEigfIgEvErrgssNfimomiig'rtriznyglg Aliirp of the normal human `diploid cells by direct transfer at B DI Mo A STRAIN OF BABIES AT DIFFERENT PASSAGE LEVELS each transfer stage of infected .human d1plo1d calls from IN iiDCs a preceding passage 1n said series 4to the next succeeding Antibody meri!) monkey Sem non-infected culture of diploid cells to provide a live Vaccine obtained rabies virus strain adapted to infect at least about 90% of Number of Virus Baby mouse Before mmm 14 days am! the cells in a non-infected sterile culture of the normal animals passage LD50 used for zation iiist inoculahuman diploid Cells. tos/ted llgns mlmumzwou* m0 ofvaccme 2. The method according to claim 1 wherein the number of transfer stages in said series of subculture pas- 0 4X1@ 55 sages isat least about 10.

1;2 1: 2 3. The method according to claim 1 wherein the num- 47 4x10, 11522558 ber of transfer stages is iii the range of from about 45 to 1:2 1z250 about 50 .Vaccine inoculated on 1st and 5th dw 4. The method according to claim 1 wherein said "CVS Virus #1,000 LDnfor mouse inoculum. adapted live rabies virus strain produced in said series of Results of the test indicate the presence of a large consubculture passages is propagated by transferring cell-free :entration of neutralizing antibodies rin sera obtained from medium from the last infected human diploid cell culture he 3 monkeys vaccinated with the HDCS-adapted virllS dispersion in said series to a non-infected sterile culture ind apparent absence of detectable antibodies in sera of of Said human diploid como he animals injected With the egg-adapted VYUS- 5. The method according to claim 1 wherein said Example XU adapted live rabies virus strain produced in said series of subculture passages subsequently is inactivated. To determine the immunizing properties of inactivated 6. The method according to daim 5 wherein said im Iabies vaccines prepared with rabies virus strains adapted o grow in HDCS in accordance with the invention, l preparations (following the procedure of Example II) I'We fables Vlfus .ll/h betaPfQPlOlaftOneif the CVS strain at different passage levels in HDCS and 7 As all ann-fables VaCClIle, 'flsslle Cllltlll' flllld COH- l preparation of the PM strain at the 12th passage in taining the adapted vii-us prepared according to the methf-IDCS were inactivated. Infected tissue culture prepara` 0d of claim 1.

ions were inactivated either by the addition of phenol at 8. As an anti-rabies vaccine, tissue culture fluid conactivation is carried out by a treatment of said adapted 13 14 taining the. adapted virus prepared according to `the meth- Peck et al.: J. American Medical Association, V01. 162 od of Iclaim 5. No. 15, Dec. 8, 1956, pp. 1373-1376.

References Cited Hayck et al.: Experimental Cell Research, v01. 25, pp. RICHARD L' HUFF Pnmary Exammer' 604-606 and 616-619, 1961. 5 LEWS GOTTS, Assistant Examiner.

Koprowski et ral.: I. Immunology, v01. 60, No. 4, pp. 533, 537 and 550-553, 1948. 

